Transient kinetic analysis for studying ionizations in RNA modification enzyme mechanisms.

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.

Best practices to ensure robust investigation of circular RNAs: pitfalls and tips.

Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs (circRNAs) that have covalently linked ends. Most mature circular RNAs are expressed at low levels, but some have known physiological functions and/or accumulate to higher levels than their associated linear mRNAs. These observations have sparked great interest into this class of previously underappreciated RNAs and prompted the development of new experimental approaches to study them, especially methods to measure or modulate circular RNA expression levels. Nonetheless, each of these approaches has caveats and potential pitfalls that must be controlled for when designing experiments and interpreting results. Here, we provide practical advice, tips, and suggested guidelines for performing robust identification, validation, and functional characterization of circular RNAs. Beyond promoting rigor and reproducibility, these suggestions should help bring clarity to the field, especially how circular RNAs function and whether these transcripts may sponge microRNAs/proteins or serve as templates for translation.

Ending on a high note: Downstream ORFs enhance mRNA translational output.

Eukaryotic mRNAs were long thought to only translate a single protein product, but it is now recognized that many mRNAs can also encode small open reading frames (ORFs). In this issue of The EMBO Journal, Wu et al characterized small ORFs in the 3' untranslated regions (3' UTRs) of human and zebrafish mRNAs and found that many are indeed translated. The peptides encoded by these downstream ORFs (dORFs) are often poorly conserved across evolution, but many dORFs are nonetheless functional, as the act of their translation can promote translation of the canonical ORF.

MicroRNA-29a inhibits glioblastoma stem cells and tumor growth by regulating the PDGF pathway.

BACKGROUND AND PURPOSE: microRNAs are small noncoding RNAs that play important roles in cancer regulation. In this study, we investigated the expression, functional effects and mechanisms of action of microRNA-29a (miR-29a) in glioblastoma (GBM). METHODS: miR-29a expression levels in GBM cells, stem cells (GSCs) and human tumors as well as normal astrocytes and normal brain were measured by quantitative PCR. miR-29a targets were uncovered by target prediction algorithms, and verified by immunoblotting and 3' UTR reporter assays. The effects of miR-29a on cell proliferation, death, migration and invasion were assessed with cell counting, Annexin V-PE/7AAD flow cytometry, scratch assay and transwell assay, respectively. Orthotopic xenografts were used to determine the effects of miR-29a on tumor growth. RESULTS: Mir-29a was downregulated in human GBM specimens, GSCs and GBM cell lines. Exogenous expression of miR-29a inhibited GSC and GBM cell growth and induced apoptosis. miR-29a also inhibited GBM cell migration and invasion. PDGFC and PDGFA were uncovered and validated as direct targets of miR-29a in GBM. miR-29a downregulated PDGFC and PDGFA expressions at the transcriptional and translational levels. PDGFC and PDGFA expressions in GBM tumors, GSCs, and GBM established cell lines were higher than in normal brain and human astrocytes. Mir-29a expression inhibited orthotopic GBM xenograft growth. CONCLUSIONS: miR-29a is a tumor suppressor miRNA in GBM, where it inhibits cancer stem cells and tumor growth by regulating the PDGF pathway.

5'-End sequencing in Saccharomyces cerevisiae offers new insights into 5'-ends of tRNAHis and snoRNAs.

tRNAHis guanylyltransferase (Thg1) specifies eukaryotic tRNAHis identity by catalysing a 3'-5' non-Watson-Crick (WC) addition of guanosine to the 5'-end of tRNAHis . Thg1 family enzymes in Archaea and Bacteria, called Thg1-like proteins (TLPs), catalyse a similar but distinct 3'-5' addition in an exclusively WC-dependent manner. Here, a genetic system in Saccharomyces cerevisiae was employed to further assess the biochemical differences between Thg1 and TLPs. Utilizing a novel 5'-end sequencing pipeline, we find that a Bacillus thuringiensis TLP sustains the growth of a thg1Δ strain by maintaining a WC-dependent addition of U-1 across from A73 . Additionally, we observe 5'-end heterogeneity in S. cerevisiae small nucleolar RNAs (snoRNAs), an observation that may inform methods of annotation and mechanisms of snoRNA processing.

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis.

The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using S-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m¹G9 or m¹A9-specific enzymes, while others are bifunctional enzymes catalyzing both m¹G9 and m¹A9. This variability is particularly intriguing given different chemical properties of the target N1 atom of guanine and adenine. Here we performed an extensive kinetic and mutational analysis of the m¹G9 and m¹A9-catalyzing Trm10 from Thermococcus kodakarensis to gain insight into the active site that facilitates this unique bifunctionality. These results suggest that the rate-determining step for catalysis likely involves a conformational change to correctly position the substrate tRNA in the active site. In this model, kinetic preferences for certain tRNA can be explained by variations in the overall stability of the folded substrate tRNA, consistent with tRNA-specific differences in metal ion dependence. Together, these results provide new insight into the substrate recognition, active site and catalytic mechanism of m¹G/m¹A catalyzing bifunctional enzymes.

Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.

Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.

Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.

Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes.